The present invention relates to a direct, very mild, efficient method of synthesis of derivatives of polyethylene glycol having one or more terminal aldehyde groups and the use of these derivatives as reagents for modifying peptides or proteins and for the preparation of other types of functionalized polyethylene glycols.
In the last decade, enormous progress in recombinant DNA technology enabled discovery and/or production of a large number of physiologically active proteins, many of them having unforeseen potential to be used as biopharmaceuticals. Unfortunately, most of these peptides and proteins exhibit very fast plasma clearance, thus requiring frequent injections to ensure steady pharmaceutically relevant blood levels of a particular peptide or protein with pharmacological activity. Further, many pharmaceutically relevant peptides and proteins, even those having human primary structure, can be immunogenic, giving rise to production of neutralizing antibodies circulating in the bloodstream. This is especially true for intravenous and subcutaneous administration, which is of particular concern for most peptide and protein drugs.
To solve these problems, various hydrophilic macromolecular compounds have been conjugated with peptide and protein drugs. This has proven to be very efficient and useful in decreasing the immunogenicity and increasing the circulatory half-life of peptide and protein drugs. Among hydrophilic macromolecular compounds, polyethylene glycol derivatives have been used most frequently for synthesis of peptide and protein conjugates, because such derivatives are non-immunogenic and very hydrophilic, and thus do not affect the three dimensional structure (folding) of protein drugs. Moreover, dynamic polyethylene chains also provide protection against hydrolytic degradation by proteolytic enzymes.
To achieve significant level of modification of peptides and proteins with polyethylene glycol (PEG) derivatives, several methods of activation have been widely used, such as the triazine method (F. F. Davis et al., U.S. Pat. No. 4,179,337), the active ester method with N-hydroxysuccinimide (F. Veronese et al., U.S. Pat. No. 5,286,637), and the direct activation method with carbonyldiimidazole (G. S. Bethell et al., 254 J. Biol. Chem. 2572-2574 (1979)). Typically, these activated PEG derivatives contain an electrophilic center that is available for reaction with nucleophilic centers on peptides and proteins, such as amino groups. The common disadvantage of these derivatives is instability in aqueous media against nonspecific hydrolysis at alkaline pH, where the modification reaction usually takes place. Recently, attachment of PEG chains to peptides and proteins via reductive alkylation has been described (P. Wirth et al., 19 Bioorg. Chem. 133-142 (1991); S. M. Chamow et al., 5 Bioconjugate Chem 133-140 (1994); O. B. Kinstler et al., 13 Pharm. Res. 996-1002 (1996)). This method requires introduction of an aldehyde group at the end of a monomethoxypolyethylene glycol (MePEG) chain that originally carried a hydroxyl group. Contrary to other activated PEG derivatives mentioned above, PEG-aldehydes, like aldehydes in general, are mostly inert to water and react selectively with the amino groups of peptides and proteins as nucleophiles in aqueous media. These two properties are highly desirable, not only because of stability upon long-term storage, but also because of high selectivity for coupling of these aldehyde-PEG derivatives and for simplicity.
Originally, MePEG was oxidized with active MnO2 to yield MePEG-ethanal (acetaldehyde) as described in U.S. Pat. No. 4,002,531 for attaching PEG chains to enzymes and other proteins. This procedure was later shown to be very inefficient by M. S. Paley and J. M. Harris, 25 J. Polym. Sci. Polym. Chem. Edn. 2447-2454 (1987). Thus, other oxidative methods, such as the Moffatt procedure, were introduced as described by Harris et al., 22 J. Polym. Sci. Polym. Chem. Edn. 341-352 (1984). These oxidative reactions, however, are not quantitative and may be accompanied by unwanted side reactions and/or MePEG chain cleavage. Further, product purification is difficult. In parallel, a more gentle and quantitative alkylation method using xcex2-bromopropionaldehyde has been used to prepare MePEG-aldehyde derivatives (J. M. Harris and M. R.-Sedaghat-Herati, U.S. Pat. No. 5,252,714). A terminal ethyl spacer was introduced to increase the stability of MePEG aldehyde derivatives in water in the presence of base since it was argued, on the basis of CH3xe2x80x94Oxe2x80x94CH2xe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94CHO chemical stability data (M. S. Paley and J. M. Harris, 25 J. Polym. Sci. Polym. Chem. Edn. 2447-2454 (1987)), that MePEG-ethanal derivatives are unstable in water in the presence of base. To the contrary, MePEG-ethanal derivatives were successfully used for protein modification when used in 20 to 200 molar excess (PEG derivative/Protein molar ratio) at pH 7.0-8.0 (P. Wirth et al., 19 Bioorg. Chem. 133-142 (1991); S. M. Charnow et al., 5 Bioconjugate Chem 133-140 (1994); O. B. Kinstler et al., 13 Pharm. Res. 996-1002 (1996)).
A new catalytic, oxidative method for conversion of alcohols into carbonyl compounds utilizing oxygen or even air as the ultimate and stoichiometric oxidant, producing water as the only byproduct, has been recently discovered and described by I. E. Marko et al., 274 Science 2044-2046(1996). The active catalyst appears to be heterogeneous, absorbed on insoluble K2CO3, and is composed of CuCl, diethylazodicarboxylate or the corresponding hydrazine, and 1,10-phenanthroline. Apolar solvents such as benzene or toluene are required. K2CO3, besides its role as a solid support, also acts as a base and as a water scavenger, but can be replaced by 4 xc3x85 molecular sieves and a catalytic amount of nonoxidizable base, such as KOBut. This process is very efficient under mild conditions (temp. 70-90xc2x0 C.), providing high degrees of conversion (80-100%), and is not only economically viable but it is also environmentally friendly.
In view of the foregoing, it will be appreciated that providing an efficient, gentle method for preparation of polyethylene glycol aldehyde derivatives would be a significant advancement in the art.
An object of the present invention is to provide a method of preparation of PEG-dialdehydes and alkoxyPEG (i.e., RO-PEG) aldehydes and their homologues that retain reactivity in water and selectively react with amino groups.
Another object of the invention is to provide PEG-dialdehydes and RO-PEG-aldehydes and their homologuestat react selectively with amino groups and are stable in water.
Yet another object of the invention is to provide bifunctional PEG and monofunctional RO-PEG derivatives and their homologues that can be prepared starting from PEG-dialdehydes and RO-PEG-aldehydes or their homologues.
Still another object of this invention is the process of preparation of bifunctional PEG and monofunctional RO-PEG derivatives and their homologues starting from PEG-dialdehydes and RO-PEG-aldehydes or their homologues.
These and other objects can be addressed by providing compositions, methods of making thereof, and methods of using thereof. An illustrative embodiment of the invention relates to the oxidation of polyethylene glycol derivatives having the formula: 
wherein R1 represents lower alkyl, R2 represents lower alkyl or H, n is an integer from about 3 to about 500, and k and m are integers from about 2 to about 12, utilizing a catalytic procedure that provides high yields of corresponding PEG-dialdehyde derivatives and RO-PEG-aldehyde derivatives having the formula: 
and enables their preparation and isolation in the absence of water.
Another illustrative embodiment of the invention relates to aldehyde derivatives of polyethylene glycos of the formula: 
wherein R1 represents lower alkyl, R2 represents lower alkyl or H, n is an integer from about 3 to about 500, and k and m are integers from about 4 to about 12.
Still another embodiment of the invention relates to polyethylene glycol derivatives of the formula: 
wherein R1 represents lower alkyl, R2 represents lower alkyl or H, n is an integer from about 3 to about 500, and k and m are integers from about 2 to about 12.
Yet another illustrative embodiment of the invention relates to polyethylene glycol derivatives of formula: 
wherein R1 represents lower alkyl, R2 represents lower alkyl or H, n is an integer from about 3 to about 500, k and m are integers from about 2 to about 12, and p is an integer from about 2 to about 12.
A further illustrative embodiment of the invention relates to methods of preparation of the polyethylene glycol derivatives summarized above. Thus, PEG-dihydrazine derivatives and RO-PEG-hydrazine derivatives are prepared by reacting a particular PEG-dialdehyde derivative or RO-PEG-aldehyde derivative with hydrazine in the presence of reducing agent such as NaBH3CN or NaBH4. Also, PEG-dithiol derivatives and RO-PEG-thiol derivatives can be synthesized from a particular PEG-dialdehyde derivative or RO-PEG-aldehyde derivative and selected mercaptoalkylamine in the presence of reducing agent such as NaBH3CN or NaBH4.
A still further embodiment of the invention relates to branched polyethylene glycol derivatives of the formula: 
wherein R1 represents lower alkyl, R2 represents lower alkyl or H, n is an integer from about 3 to about 500, k and m are integers from about 2 to about 12, and r is an integer from 0 to about 5. These branched polyethylene glycol derivatives are prepared by reacting a particular RO-PEG-aldehyde derivative with a selected diaminocarboxylic acid, which does not necessarily have to be an xcex1-acarboxylic acid as shown in the formula but also can be xcex2-, xcex3-, or xcex4-carboxylic acid, in the presence of reducing agent such as NaBH3CN or NaBH4.
Another illustrative embodiment of the invention relates to branched polyethylene glycol derivatives of the formula: 
wherein R1 represents lower alkyl, R2 represents lower alkyl or H, n is an integer from about 3 to about 500, k and m are integers from about 2 to about 12, r is an integer from 0 to about 5, and s is 2 or 3. This type of branched polyethylene glycol derivative is prepared starting from monoaminoPEG derivatives, as set forth below, and reacting them with dicarboxylic acid cyclic anhydrides, thus converting monoaminoPEG derivatives into monocarboxyPEG derivatives. A selected activated monocarboxyPEG derivative is then reacted with a selected diaminocarboxylic acid, which does not necessarily have to be an a-carboxylic acid but can also be xcex2-, xcex3-, or xcex4-carboxylic, yielding a branched polyethylene glycol derivative as specified by the general formula above.
Finally, another illustrative embodiment of the invention relates to methods of preparation of amino-PEG derivatives of the formula: 
wherein R1 represents lower alkyl, R2 represents lower alkyl or H, n is an integer from about 3 to about 500, and k and m are integers from about 3 to about 12. These derivatives can be synthesized by reductive amination of corresponding PEG-dialdehyde derivatives or RO-PEG-aldehyde derivatives, prepared as disclosed above, with ammonium salt in the presence of reducing agent such as NaBH3CN or NaBH4.